Purpose: To explore the perspectives of people with hip and knee arthritis regarding a physiotherapy extended-role practitioner (ERP) model of care in a rural setting. Method: Using semi-structured interviews, a qualitative descriptive case study was undertaken with 13 participants from a rural family practice located in the province of Ontario, Canada, who had all been assessed by an ERP. Transcribed interviews were analyzed for emergent themes. Results: Three main themes were identified: (1) timely access to care, (2) distance as a factor in seeking care, and (3) perceptions of the ERP model of care. Conclusions: Participants reported many positive experiences with the physiotherapy ERP rural model. Processes related to minimizing travel required to access care are important for those in rural communities. An ERP model of care offers competent care that includes musculoskeletal diagnosis as well as time for educating patients and addressing questions.
access to health care; arthroplasty; rural health; accès aux soins de santé; arthroplastie; santé rurale; spécialité de la physiothérapie
Chronic lymphocytic leukaemia (CLL) patients have an increased risk of other malignancies. This may be due to surveillance bias, treatment or immunosuppression.
Cohort study of 612 consecutively diagnosed CLL patients in a Canadian province, with comparisons to follicular lymphoma (FL) patients.
Treated CLL patients had a 1.7-fold increased risk of second cancers compared with untreated CLL patients. As compared with untreated FL patients, untreated CLL patients had a two-fold increased incidence of second malignancies.
Chronic lymphocytic leukaemia patients have an inherent predisposition to second cancers and the incidence is further increased by treatment.
chronic lymphocytic leukaemia; follicular lymphoma; epidemiology; second malignancies; non-melanoma skin cancers; treatment
HIV and Non communicable diseases (NCDs) are major problem of public health importance in developing countries. This study was conducted to explore and establish information on the magnitude, distribution of NCDs risk factors among people living with HIV (PLWHIV) which is scarce in Tanzania.
A cross sectional study was conducted to PLWHIV from 12 care and treatment clinics in Dar es Salaam and Mbeya regions from October 2011 to February 2012. Data on demographic characteristics, NCD risk factors including behavioral, biochemical tests and physical measurements was collected from PLWHIV.
Of 754 PLWHIV recruited, 671(89.0%) consented to participate in the study and 354/671(52.8%) were on antiretroviral therapy (ART). The following NCD risk factors: raised blood levels of low density lipoprotein (61.3% vs 38.7%, p < 0.001) total cholesterol (TC) (71.6% vs 28.4%, p < 0.001) and triglyceride (67.0% vs 33.0%, p = 0.001) as well as overweight/obesity (61.1% vs 38.9%, p = 0.010), abnormal waist circumference (61.7% vs 38.3%, p < 0.001) and being aged >40 years (63.3% vs 36.7%, p < 0.001) were more prevalent among PLWHIV on ART than ART naïve. The prevalence of Diabetes mellitus among PLWHIV was 4.2% and was slightly high among those ART naïve (4.7% vs 3.7%). The prevalence of hypertension was 26.2% and was high among those on ART (30.0% vs 21.9%, p = 0.010). Being aged >40 years (AOR = 2.52, 95% CI 1.37-4.63), abnormal waist circumference (AOR = 2.37 95% CI 1.13-5.00), overweight/obesity (AOR = 2.71, 95% CI 1.26-5.84) and male sex (AOR = 1.17, 1.02-4.20) were the predictors of hypertension among patients on ART while raised TC (AOR = 1.47 (1.01-2.21) and being aged >40 years (AOR = 3.42, 95% CI 2.06-5.70) were predictors for hypertension among ART naïve patients.
This study has revealed that the magnitude of NCD risk factors is significantly higher among PLWHIV on ART than those not on ART. Initiating and strengthening of interventions for minimizing preventable NCD risks should be considered when initiating ART among PLWHIV. Regular monitoring of NCD risk factors is of paramount importance among ART patients.
Non-communicable diseases; Risk factors; ART; People living with HIV; Tanzania
Fludarabine, a nucleoside analogue, is commonly used in combination with other agents for the treatment of chronic lymphocytic leukaemia (CLL). In previous studies, valproic acid (VPA), an inhibitor of histone deacetylases, combined with fludarabine to synergistically increase apoptotic cell death in CLL cells. In the present study, we found that the combination of fludarabine and VPA decreases the level of the anti-apoptotic proteins Mcl-1 and XIAP in primary CLL cells. Treatment with fludarabine alone, or in combination with VPA, led to the loss of lysosome integrity, and chemical inhibition of the lysosomal protease cathepsin B, using CA074-Me, was sufficient to reduce apoptosis. VPA treatment increased cathepsin B levels and activities in primary CLL cells, thereby priming CLL cells for lysosome-mediated cell death. Six previously treated patients with relapsed CLL were treated with VPA, followed by VPA/fludarabine combination. The combined therapy resulted in reduced lymphocyte count in five out of six and reduced lymph node sizes in four out of six patients. In vivo VPA treatment increased histone-3 acetylation and cathepsin B expression levels. Thus, the synergistic apoptotic response with VPA and fludarabine in CLL is mediated by cathepsin B activation leading to a decrease in the anti-apoptotic proteins.
CLL; VPA; HDAC; cathepsin; lysosome
Free-flying honey bees (Apis mellifera L.) reactions were observed when presented with varying schedules of post-reinforcement delays of 0 s, 300 s, or 600 s. We measured inter-visit-interval, response length, inter-response-time, and response rate. Honey bees exposed to these post-reinforcement delay intervals exhibit one of several patterns compared to groups not encountering delays, and had longer inter-visit-intervals. We observed no group differences in inter-response time. Honey bees with higher response rates tended to not finish the experiment. The removal of the delay intervals increased response rates for those subjects that completed the trials.
We previously reported that children in the UKALL XI ALL trial with HLA-DP 1 and -DP 3 supertypes had significantly worse event-free survival (EFS) than children with other DP supertypes. As DP 1 and DP 3 share two of four key antigen-binding amino-acid polymorphisms (aspartic acid84–lysine69), we asked whether Asp84-Lys69 or Asp84 alone were independent prognostic indicators in childhood acute lymphoblastic leukemia (ALL). We analysed EFS in 798 UKALL XI patients, stratified by Asp84-Lys69 vs non-Asp84-Lys69, for a median follow-up of 12.5 years. Asp84-Lys69 was associated with a significantly worse EFS than non-Asp84-Lys69 (5-year EFS: Asp84-Lys69: 58.8% (95% CI (confidence of interval): 52.7–64.9%); non-Asp84-Lys69: 67.3% (63.4–71.2%); 2P=0.007). Post-relapse EFS was 10% less in Asp84-Lys69 than non-Asp84-Lys69 patients. EFS was significantly worse (P=0.03) and post-relapse EFS marginally worse (P=0.06) in patients with Asp84 compared with Gly84. These results suggest that Asp84-Lys69 predicted adverse EFS in the context of UKALL XI because of Asp84, and may have influenced post-relapse EFS. We speculate that this may be due to the recruitment of Asp84-Lys69-restricted regulatory T cells in the context of this regimen, leading to the re-emergence of residual disease. However, functional and molecular studies of the prognostic value of this and other HLA molecular signatures in other childhood ALL trials are needed.
childhood ALL; HLA-DP supertype; DP molecular signature; event-free survival; relapse
The phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is well known for the regulation of cell survival, proliferation, and some metabolic routes.
Meterials and Methods
In this study, we document a novel role for the PI3-K/Akt pathway during cell death induced by apoptin, a tumour-selective inducer of apoptosis.
We show for the first time that apoptin interacts with the p85 regulatory subunit, leading to constitutive activation of PI3-K. The inhibition of PI3-K activation either by chemical inhibitors or by genetic approaches severely impairs cell death induced by apoptin. Downstream of PI3-K, Akt is activated and translocated to the nucleus together with apoptin. Direct interaction between apoptin and Akt is documented. Co-expression of nuclear Akt significantly potentiates cell death induced by apoptin. Thus, apoptin-facilitated nuclear Akt, in contrast to when in its cytoplasmic pool, appears to be a positive regulator, rather than repressor of apoptosis.
Our observations indicate that PI3-K/Akt pathways have a dual role in both survival and cell death processes depending on the stimulus. Nuclear Akt acts as apoptosis stimulator rather than as a repressor, as it likely gains access to a new set of substrates in the nucleus. The implicated link between survival and cell death pathways during apoptosis opens new pharmacological opportunities to modulate apoptosis in cancer, for example through the manipulation of Akt’s cellular localization.
PMID: 18021174 CAMSID: cams687
Intensive insulin therapy (IIT) for glycemic control in critically ill patients has been shown to be beneficial. Continuous glucose monitoring systems (CGMSs) have been approved as an adjunct to complement standard glucose monitoring in type 2 diabetes mellitus. This study was designed to evaluate the accuracy of a real-time CGMS (DexComTM STS) in the intensive care unit (ICU). We also evaluated its reliability and accuracy using a hyperinsulinemic-euglycemic and a hyperglycemic clamp study.
Nineteen patients were enrolled in this 7-day study [13 = surgical intensive care unit (SICU), 6 = burn intensive care unit (BICU)]. The patients were on IIT for at least 2 h prior the subcutaneous sensor insertion. Mean age and body mass index for SICU and BICU patients were 60.3 ± 3.7 and 64.5 ± 6.2 years and 36.6 ± 5.0 and 33.85 ± 3.4 kg/m2, respectively. DexCom accuracy was analyzed separately for the Johnson & Johnson (J&J) calibration finger sticks, Roche Accucheck finger sticks, and the Hitachi 917 analyzer measurements on serum using Clarke error grid analysis and Bland–Altman analysis. In the clamp studies, 20 patients were enrolled, and the data were analyzed similarly.
There were 1065 pairs of DexCom–Accucheck, 232 pairs of DexCom–J&J, and 84 pairs of DexCom–Hitachi in ICU patients. For DexCom–Accucheck, 68.26% of the pairs fell into zone A, 31.83% into zone B, and 0.75% into zone C. There were no values in zones D or E.
From the 1102 matching DexCom–Beckman pairs in clamp studies, 42.29% were in zone A, 55.90% were in zone B, and 4.08% were in zone C.
Despite the high percentage of measurements in zones A and B, underestimation of hypoglycemia by DexCom measurements makes it an unreliable device in the ICU setting.
continuous glucose monitors; DexCom; diabetes; glycemic control; intensive care unit; intensive insulin therapy
Background: Deoxycytidine kinase (dCK) is responsible for the activation of several clinically important deoxynucleoside analogues used for the treatment of haematological and solid malignancies.
Aim: To measure dCK expression in tumour cells from different origins.
Method: A rabbit antihuman dCK antibody was used for the immunocytochemical detection of dCK expression in three leukaemic cell lines (HL60, U937, and CCRF-CEM) and 97 patient samples (paediatric acute myeloid leukaemia (AML) and lymphoid leukaemia (ALL), retinoblastoma, paediatric brain tumours, and adult non-small cell lung cancer (NSCLC)).
Results: CCRF-CEM, U937, and HL60 cells stained positively for dCK and the degree of expression correlated with dCK activity. dCK expression varied between tumour types and between individual patients within one tumour type. dCK was located predominantly in the cytoplasm. The staining intensity was scored as negative (0), low (1+), intermediate (2+), or high (3+). Expression of dCK was high in AML blasts. In contrast, brain tumour samples expressed low amounts of dCK. dCK staining ranged from low (1+) to high (3+) in ALL blasts, retinoblastoma, and NSCLC tissue samples. Staining was consistent (interobserver variability, 88%; κ = 0.83) and specific. Western blotting detected the dCK protein appropriately at 30 kDa, without additional bands.
Conclusions: Immunocytochemistry is an effective and reliable method for determining the expression of dCK in patient samples and requires little tumour material. This method enables large scale screening of dCK expression in tumour samples.
deoxycytidine kinase; immunocytochemistry; childhood acute leukaemia; solid tumours; deoxynucleoside analogues
Cytarabine (ara-C) is the most effective agent for the treatment of acute myeloid leukaemia (AML). Aberrant expression of enzymes involved in the transport/metabolism of ara-C could explain drug resistance. We determined mRNA expression of these factors using quantitative-real-time-PCR in leukemic blasts from children diagnosed with de novo AML. Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P=0.007). In vitro sensitivity to deoxynucleoside analogues was determined using the MTT-assay. Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (rp=−0.46; P=0.001), with three-fold lower hENT1 mRNA levels in resistant patients (P=0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC50 values of cladribine (rp=−0.30; P=0.04), decitabine (rp=−0.29; P=0.04) and gemcitabine (rp=−0.33; P=0.02). Deoxycytidine kinase (dCK) and cytidine deaminase (CDA) mRNA expression seemed to correlate with in vitro sensitivity to gemcitabine (rp=−0.31; P=0.03) and decitabine (rp=0.33; P=0.03), respectively. The dCK/PN-I ratio correlated inversely with LC50 values for gemcitabine (rp=−0.45, P=0.001) and the dCK/CDA ratio seemed to correlate with LC50 values for decitabine (rp=−0.29; 0.04). In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML.
childhood acute myeloid leukaemia; deoxynucleoside analogues; cytarabine; hENT1
AIMS—To examine the clinical and
biological features of acute lymphoblastic leukaemia in children with
Down's syndrome (DS), to compare their survival with other children,
and to determine if entry to trials and survival has improved.
METHODS—Examination of presenting
features and response to treatment in patients treated in two
consecutive national trials, MRC UKALL X and XI.
RESULTS—The proportion of children
with DS was significantly higher in UKALL XI (1.9%) than UKALL X
(0.9%). Children with DS tended to be under 10 years and to have the
common ALL subtype. Cytogenetic analysis showed that favourable
features, such as high hyperdiploidy and t(12;21) were less frequent
but also that there was a lack of translocations associated with a poor
prognosis. Children with DS showed no increase in risk of relapse at
any site but their survival and event free survival were inferior to
other children. These results were caused by an increased number of
infective deaths during remission (11% compared to 2%). At five years
overall survival was 73% in DS children compared with 82% in other
children; event free survival was 53% compared to 63% in non-DS children.
CONCLUSIONS—Entry of children with
DS to national trials has increased and survival has improved. However
they remain at risk of relapse and also of treatment related mortality.
These findings emphasise the need for both intensive chemotherapy and
optimal supportive care.
Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate α-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with α2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization—time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing α2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.
To determine whether pentoxifylline 400 mg (Trental 400) taken orally three times daily, in addition to ambulatory compression bandages and dressings, improves the healing rate of pure venous ulcers.
Randomised, double blind placebo controlled trial, parallel group study of factorial design, permitting the simultaneous evaluation of alternative pharmaceutical, bandaging, and dressings materials.
Leg ulcer clinics of a teaching and a district general hospital in southern Scotland.
200 patients with confirmed venous ulcers and in whom other major causal factors were excluded.
Pentoxifylline 400 mg three times daily or placebo.
Main outcome measure
Complete healing (full epithelialisation) of all ulcers on the trial leg.
Complete healing occurred in 65 of the 101 (64%) patients receiving pentoxifylline and 52 of the 99 (53%) patients receiving placebo.
The difference in the healing rates between patients taking pentoxifylline and those taking placebo did not reach statistical significance.
Key messagesLeg ulcers cost the NHS around £400 million per annum50%-75% of venous leg ulcers can be succesfully treated with dressings and compression bandages but take many months to healA drug that reduced the healing time of venous ulcers would be useful, although no agent has been proved to be effective to dateTrials with pentoxifylline, a vasoactive drug used in the treatment of peripheral vascular diseases, as an adjunct to the treatment of venous ulcers have been inconclusiveAt the 5% level, pentoxifylline had a non-significant effect on healing rates of pure venous ulcers
BACKGROUND: Nosocomial aspergillosis is a well known complication of immunosuppression in cancer patients and those undergoing transplantation and has usually been associated with major building construction or demolition. An observational study is reported of the hospital environment associated with an outbreak of aspergillosis in a paediatric oncology ward. METHODS: All cases of aspergillosis were identified from the hospital records and categorised as definite or probable according to the extent of supportive clinical and laboratory findings. All relevant aspects of building ventilation, air filtration, and aerosol generation considered relevant were examined and air samples for fungi were taken in triplicate at 25 sites using a slit sampler with appropriate culture media. RESULTS: Six cases of aspergillosis were identified over one year out of the 148 patients who attended the unit - the only part of the hospital where cases were found. Examination of the building services and function suggested that the cause or source was isolated to this paediatric oncology/haematology ward and may have been attributed to a defective disposal conduit door as well as the dispersal of a contaminated aerosol from the ward vacuum cleaner which had the highest measured concentrations of Aspergillus fumigatus in or around the building (65 colony forming units (cfu)/m3 compared with 0-6 cfu/m3 elsewhere). No further cases were identified in the two years after these hygiene arrangements were changed. CONCLUSIONS: The investigation of this outbreak of nosocomial aspergillosis identified several possible sources of fungally contaminated aerosol which could have been implicated as the cause. Their modification was followed by a reduction in the incidence of further cases. Each should be incorporated as an issue of importance in hospital building design and hygiene.
AIMS--Children in a United Kingdom national trial for relapsed non-B lymphoblastic leukaemia (ALL) had their diagnostic and relapse marrow cytomorphology compared to see what changes occur during the evolution of the disease. METHODS--Each relapse slide was assessed blindly for French American British (FAB) type and other morphological features by a panel of three independent microscopists without reference to each other or any diagnostic material. Diagnostic slides had been assessed by the same panel on an earlier occasion. RESULTS--A total of 134 consecutive children was studied. Six (5%) were classified as FAB type L2 at diagnosis, compared with 18 (13%) at relapse (a difference of 9%). Twenty two (16%) changed their FAB type, 17 (13%) from L1 to L2 and five (4%) from L2 to L1. The FAB score fell at relapse in 34 children and rose in 14, a difference of 14%. Cell size was the commonest feature to change (increasing in 22 and diminishing in nine) followed by prominent nucleoli (appearing in 21 and disappearing in six). Forty four (33%) children had vacuolated blasts at diagnosis, compared with 48 (36%) at relapse. Twenty five changed their vacuole score substantially, 14 gaining > 10% and 11 falling < 10%. CONCLUSIONS--These findings reflect the variability of lymphoblast cytomorphology, but also show a trend for cells to have more prominent nucleoli and greater size at relapse. Factors controlling these features of the FAB type are unknown, but they may simply be related to the growth fraction of a particular disease and not to any lineage specific biological feature.
Human mitochondrial trifunctional protein (TFP) is a heterooctamer of four alpha- and four beta-subunits that catalyzes three steps in the beta-oxidation spiral of long-chain fatty acids. TFP deficiency causes a Reye-like syndrome, cardiomyopathy, or sudden, unexpected death. We delineated the molecular basis for TFP deficiency in two patients with a unique phenotype characterized by chronic progressive polyneuropathy and myopathy without hepatic or cardiac involvement. Single-stranded conformation variance and nucleotide sequencing identified all patient mutations in exon 9 of the alpha-subunit. One patient is homozygous for the T845A mutation that substitutes aspartic acid for valine at residue 246. The second patient is a compound heterozygote for the T914A that substitutes asparagine for isoleucine at residue 269 and a C871T that creates a premature termination at residue 255. Allele-specific oligonucleotide hybridization studies revealed undetectable levels of the mRNA corresponding to the mutant allele carrying the termination codon. This study suggests a novel genotype-phenotype correlation in TFP deficiency; that is, mutations in exon 9 of the alpha-subunit, which encodes a linker domain between the NH2-terminal hydratase and the COOH-terminal 3-hydroxyacyl-CoA dehydrogenase, result in a unique neuromuscular phenotype.
We have undertaken a study to investigate the contribution of the htrB gene to the virulence of pathogenic Salmonella typhimurium. An htrB::mini-Tn10 mutation from Escherichia coli was transferred by transduction to the mouse-virulent strain S. typhimurium SL1344 to create an htrB mutant. The S. typhimurium htrB mutant was inoculated into mice and found to be severely limited in its ability to colonize organs of the lymphatic system and to cause systemic disease in mice. A variety of experiments were performed to determine the possible reasons for this loss of virulence. Serum killing assays revealed that the S. typhimurium htrB mutant was as resistant to killing by complement as the wild-type strain. However, macrophage survival assays revealed that the S. typhimurium htrB mutant was more sensitive to the intracellular environment of murine macrophages than the wild-type strain. In addition, the bioactivity of the lipopolysaccharide (LPS) of the htrB mutant was reduced compared to that of the LPS from the parent strain as measured by both a Limulus amoebocyte lysate endotoxin quantitation assay and a tumor necrosis factor alpha bioassay. These results indicate that the htrB gene plays a role in the virulence of S. typhimurium.
The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A beta-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV(A), which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a well-defined murine typhoid model of animal pathogenesis. To this end, an r- m+ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter- and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.
To define the role of the surface lipooligosaccharide (LOS) of Haemophilus ducreyi in the pathogenesis of chancroid, Tn916 mutants of H. ducreyi 35000 defective in expression of the murine monoclonal antibody (MAb) 3F11 epitope on H. ducreyi LOS were identified by immunologic screening. One mutant, designated 1381, has an LOS which lacks the MAb 3F11 epitope and migrates with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene disrupted by the Tn916 element in strain 1381 was identified by cloning the sequences flanking the Tn916 element. The sequences were then used to probe a lambda DASHII genomic library. In strain 1381, Tn916 interrupts a gene which encodes an open reading frame (ORF) with an Mr of 40,246. This ORF has homology to the product of the rfaK gene of Escherichia coli. The major LOS glycoform produced by strain 1381 was analyzed by using a combination of mass spectrometry, linkage and composition analysis, and 1H nuclear magnetic resonance spectroscopy. The major LOS species was found to terminate in a single glucose attached to the heptose (L-glycero-D-manno-heptose, or Hep) trisaccharide core. In the wild-type strain 35000, glucose serves as the acceptor for the addition of the D-glycero-D-manno-heptose (or DDHep), which extends to form the mature branch of the H. ducreyi LOS. This mature oligosaccharide is in turn partially capped by the addition of sialic acid (NeuAc), i.e., NeuAc2 alpha-->3Gal beta1-->4GlcNAc beta1-->3Gal beta1-->4DDHep alpha1-->6Glc beta1 (W. Melaugh et al., Biochemistry 33:13070-13078, 1994). Since this LOS terminates prior to the addition of the branch DD-heptose, this gene is likely to encode the D-glycero-D-manno-heptosyltransferase. Strain 1381 exhibits a significant reduction in adherence to and invasion of primary human keratinocytes. This defect was complemented by the cloned heptosyltransferase gene, indicating that the terminal portion of the LOS oligosaccharide plays an important role in adherence to human keratinocytes.
Haemophilus influenzae is an important human pathogen. The lipooligosaccharide (LOS) of H. influenzae has been implicated as a virulence determinant. To better understand the assembly of LOS in nontypeable H. influenzae (NtHi), we have cloned and characterized the rfaD and rfaF genes of NtHi 2019, which encode the ADP-L-glycero-D-manno-heptose-6-epimerase and heptosyltransferase II enzymes, respectively. This cloning was accomplished by the complementation of Salmonella typhimurium lipopolysaccharide (LPS) biosynthesis gene mutants. These deep rough mutants are novobiocin susceptible until complemented with the appropriate gene. In this manner, we are able to use novobiocin resistance to select for specific NtHi LOS inner core biosynthesis genes. Such a screening system yielded a plasmid with a 4.8-kb insert. This plasmid was able to complement both rfaD and rfaF mutants of S. typhimurium. The LPS of these complemented strains appeared identical to the wild-type Salmonella LPS. The genes encoding the rfaD and rfaF genes from NtHi 2019 were sequenced and found to be similar to the analogous genes from S. typhimurium and Escherichia coli. The rfaD gene encodes a polypeptide of 35 kDa and the rfaF encodes a protein of 39 kDa, as demonstrated by in vitro transcription-translation studies. Isogenic mutants which demonstrated truncated LOS consistent with inner core biosynthesis mutants were constructed in the NtHi strain 2019. Primer extension analysis demonstrated the presence of a strong promoter upstream of rfaD but suggested only a very weak promoter upstream of rfaF. Complementation studies, however, suggest that the rfaF gene does have an independent promoter. Mass spectrometric analysis shows that the LOS molecules expressed by H. influenzae rfaD and rfaF mutant strains have identical molecular masses. Additional studies verified that in the rfaD mutant strain, D-glycero-D-manno-heptose is added to the LOS molecule in place of the usual L-glycero-D-manno-heptose. Finally, the genetic organizations of the inner core biosynthesis genes of S. typhimurium, E. coli, and several strains of H. influenzae were examined, and substantial differences were uncovered.
Mycobacterium avium secretes iron-binding siderophores called exochelins. The exochelins from M. avium have previously been reported to have unsaturated side chains that terminate in carboxylic acid. In contrast, our data show the side chains to be both saturated and unsaturated and to terminate with either a carboxylate or methyl ester.
In 1994 the Ontario government passed one of the world's toughest packages of antitobacco legislation. The Tobacco Control Act places restrictions on who can sell tobacco products, provides for severe penalties for retailers who sell to minors, bans smoking in many public places and severely restricts the use of designated smoking areas in others. The province has had antismoking legislation before, but enforcement was lax; this time enforcement of the law, particularly as it concerns retailers who sell to minors, has been given priority Brenda Gibson asks if these tough new measures are working.
The major lipooligosaccharides of the sexually transmitted pathogen Haemophilus ducreyi 35000 have been previously found to terminate in N-acetyllactosamine and sialyl-N-acetyllactosamine, Neu5Ac alpha 2-->3Gal beta 1-->4GlcNAc (W. Melaugh, N. J. Phillips, A. A. Campagnari, M. V. Tullius, and B. W. Gibson, Biochemistry 33: 13070-13078, 1994). In this study, mass spectrometry and composition analyses have shown that the lipooligosaccharides from three other H. ducreyi strains also contain N-acetyllactosamine and are highly sialylated (approximately 30 to 50%), although one African strain was found to contain neither of these structural features.